Article Figures & Data
Figures
- Figure 1. Immunoresponsiveness of cell types selected as highly susceptible to HEV infection.
(A, B, C) Quantification of transcript expression levels of representatives of the IFN-I/III–related pathway, that is, MXA and ISG15, NF-KB–induced signaling, that is, TNF and IL-6, upon stimulation by agonists of TLR3, that is, addition of polyI:C; RIG-I/MDA-5 cytosolic sensors by transfection of polyI:C LMW and addition of recombinant IFN-β, IFN-λ3, and TNF at the indicated concentrations for 6-hour incubation, determined by RT–qPCR. (A, B, C) Analyses were performed in Huh-7.5 cells (A), HepG2/C3A cells (B), and PLC3 cells (C); bars represent fold expression; means ± SD; each dot represents one independent experiment, n = 4 for treatments including polyI:C addition/transfection and IFN-β treatment, n = 3 for treatment with IFN-λ and TNF; statistical analysis was performed using the paired pairwise Wilcoxon test; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure S1. HEV-replicating cell-types and HEV mutants.
(A, B) Quantification of the replication levels of HEV at 6 d post-electroporation with 10 μg RNA [HEV cells] or mock electroporation without RNA [cont cells] (as described in the Materials and Methods section) by RT–qPCR detection of HEV RNA (amplicon/primers designed in ORF2) in HepG2/C3A (A) and PLC3 cells (B), respectively; means ± SD; each dot represents one independent experiment; n = 4 for PLC3 cells and HepG2/C3A cells; statistical analysis was done using the Wilcoxon rank-sum test with continuity correction and P-value adjustment with the Bonferroni method; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (C) HEV infection levels determined by immunodetection of ORF2 protein using 1E6 antibody are presented as representative images in HEV-producing PLC3 and HepG2/C3A cells versus control cells [cont] transfected with no RNA as a negative control, at 6 d.p.e. (D) Quantification by flow cytometry of the frequency of HEV ORF2-positive PLC3 cells. Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction and P-value adjustment with the FDR method; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (E) Illustrative table describing the differences between WT and mutants in terms of intracellular and extracellular viral products in electroporated cells.
- Figure 2. pDC response against HEV-infected cells.
(A, B) Quantification of the transcript expression levels of representatives of IFN-I/III signaling (i.e., MXA, ISG15, OAS2, and IFN-λ1) and NF-KB–related pathway (i.e., TNF and IL-6) determined at 6 d.p.e. of HepG2/C3A (A) and PLC3 (B) cells that were electroporated with 10 μg RNA [HEV cells] or mock-electroporated without RNA [cont cells], in the absence (left panels) or in co-culture with pDCs for 18 h (right panels), as indicated. No pDC and pDC co-culture conditions have been separated into left and right panels as steady-state levels of gene expression are different in these two datasets, and therefore, cross-comparisons between the two panels must be avoided. Bars represent copy number per μg total RNA; means ± SD; each dot represents one independent experiment (n = 3–8). Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (C, D) pDCs were co-cultured with HEV-electroporated PLC3 or HepG2/C3A cells for 18 and 48 h. (C, D) Quantification of IL-29/28A/28B in supernatants of pDCs co-cultured with HEV-replicating HepG2/C3A (C) and PLC3 cells (D); n ≥ 3; statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (E, F, G) pDCs were co-cultured with HEV-electroporated PLC3 cells, and their supernatants, in various settings, or treated by inhibitors, as indicated, for 18 h. (E) Quantification of IFN-α in supernatants of pDCs co-cultured with HEV-replicating PLC3 cells [HEV cells] or treated with supernatants from HEV-infected cells [HEV SN] versus in the absence of pDCs [no pDC]; the uninfected [cont] cells were electroporated without HEV RNA and used as a negative control. Bars represent means ± SD, and each dot represents one independent experiment (n = 4). (F) Quantification of IFN-α in supernatants of pDCs in co-culture or separated from HEV-electroporated PLC3 cells by a permeable membrane (0.4 μm) of transwell [transwell setting]. The TLR7 agonist, imiquimod [IMQ], was used as a positive control. (E) Results presented as in (E); n = 4. (G) Co-culture of pDCs and HEV-electroporated PLC3 cells was treated by inhibitors of TLR7 [IRS661], blocking antibodies against αLβ2-integrin and ICAM-1, followed by the quantification of IFN-α in supernatants of the co-cultures. (E) Results presented as in (E); n > 3 independent experiments. Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure 3. Control of HEV infection by pDCs.
(A, B) Quantification of HEV RNA replication levels in HepG2/C3A (A) and PLC3(B) cells, in the absence or in co-culture with pDCs, as indicated; means ± SD; each dot represents one independent experiment in terms of fold expression compared with [no pDC] condition; n = 4 for HepG2/C3A cells and PLC3 cells. Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction and P-value adjustment with the Bonferroni method; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (C) Quantification of ORF2-expressing cells in the presence or absence of pDCs by flow cytometry; fold change in HEV-replicating (ORF2+) PLC3 cells in the absence and presence of pDCs for 48 h (n = 4). (D, E) HEV-replicating PLC3 cells (GFP− ORF2+) and uninfected cells (GFP+ ORF2−) were co-cultured in the presence and absence of pDCs for 48 h. (D) Quantification of ORF2- and GFP-expressing cells by flow cytometry; (D) results are presented as representative dot blots. (E) Fold change in ORF2+ GFP− and ORF2+ GFP+ cells was quantified by flow cytometry. Bars represent means ± SD, and each dot represents one independent experiment (n = 5). Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction and P-value adjustment with the Bonferroni method; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure S2. Determinant of the production of ORF2g/c forms.
(A) Construction: a stop codon introduced in the ORF2 signal peptide without affecting ORF3 expression. (B) WB detection of ORF2 in the supernatant [SN] and cell lysates [cells] of HEV-producing PLC3 cells (using 1E6 mAb: detect all ORF2 species, mostly ORF2g/c in WT SN). (C) Immunoprecipitation in SN of all ORF2 forms versus ORF2i using P3H2 and P1H1 mAbs, respectively. (D, E). Quantification of HEV RNA levels in SN and HEV-infected PLC3 cells and extracellular infectious titers; mean ± SD; n ≥ 3. (F) Imaging of ORF2 (1E6 mAb) in Huh-7.5 cells infected by WT and STOP mutant. (G) Western blot detection of ORF2 in the SNs of HEV-replicating HepG2/C3A and PLC3 cells 6 d.p.e. with HEV WT or STOP RNA in two independent experiments (using 1E6, which detects all ORF2 species, mostly ORF2g/c in WT SN).
- Figure 4. Impact of ORF2 nuclear localization on pDC activation by studying co-cultures with specific HEV mutants.
Mutations were designed to inactivate i) nuclear translocation of ORF2 as [5R5A] mutant, ii) the ORF2 export from nucleus as [NES] mutant, and iii) ORF2g/c secretion as [STOP] mutant. Cells harboring the [GAD] mutant that is deficient for viral replication and/or uninfected [cont] cells were used as negative controls. Plasmids containing the above-mentioned mutations in the HEV genome versus WT served as templates for in vitro transcription into HEV RNA, then transfected in either HepG2/C3A or PLC3 cells, as indicated. (A, B) Intracellular distribution of ORF2 (1E6 antibody, followed by targeting secondary antibody with Alexa Fluor 488) in HepG2/C3A (A) or PLC3 (B) cells electroporated with different mutants of HEV. (C, D) Quantification of HEV replication levels of mutants versus WT in HepG2/C3A (n = 4) (C) or PLC3 cells (n = 3–6) (D). Results represent HEV RNA copy number (primer/amplicon designed in ORF2 RNA) per μg total RNA, detected by RT–qPCR; bars represent means ± SD, and dot represents independent experiments. (E, F) Quantification of IFN-α in supernatants of pDCs that were co-cultured with cells harboring HEV with the indicated mutant genome versus WT, and GAD or uninfected, as negative controls; for 18 h; 6 d.p.e. of HepG2/C3A cells (n = 5) (E) and PLC3 cells (n = 3–8) (F); bars represent means ± SD, and dots represent independent experiments. Statistical analysis was done using the Wilcoxon rank-sum test; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure S3. Differences in HEV-induced immune signaling among cell types.
(A, B) Quantification of the transcript expression levels of TNF determined at 6 d.p.e. in HepG2/C3A (A) and PLC3 (B) cells. Bars represent the copy number per μg total RNA; means ± SD; each dot represents one independent experiment (n = 3–5). Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure S4. Confocal imaging pipeline to quantify cell proximity between pDCs and infected cells.
(A) Illustrative pipeline explaining steps in acquisition and treatment of confocal images used to quantify infected cells, enumerate the contacts between infected cells and pDCs, and measure the contact area. (B) Illustration describing criteria of contact enumeration as set in the macro used to analyze the confocal images. (C) Violin plots represent the area of contact/proximity of PLC3-infected cells and pDCs from automatic tracking (i.e., defined within a cell-to-cell distance < 1 μm; contact area> 0 µm2) with detection of CTV/pDCs and CMTPX+ ORF2+/infected cells, as a reference; violin plots present the median and each dot for independent image (n > 6) from three distinct experiments. Statistical analysis was done using the Wilcoxon rank-sum test.
- Figure 5. Effect of ORF2 expression on localization on physical contacts between HEV cells and pDCs.
(A, B, C) pDCs co-cultured with HEV-replicating PLC3 cells 6 d.p.e, bearing the indicated mutations versus WT and GAD, as reference and negative control, respectively. Confocal imaging of pDCs and HEV co-culture performed after 4-h incubation. (A) Representative confocal stack imaging of ORF2 immunostaining (green) in infected PLC3 cells, which were stained with CellTracker Red before co-cultures (CMTPX, red), combined with pDCs stained with Cell-Tracer Violet before co-culture (CTV; blue). (B) Frequency of HEV-replicating (ORF2+) PLC3 cells detected among the cells defined as non-pDC (CTV−/CMTPX+); bars present means ± SD and each dot for independent image (n ≥ 8) from four distinct experiments. (C) Contact/proximity of PLC3-infected cells and pDCs with detection of CTV/pDCs and CMTPX+ ORF2+/infected cells, as reference; bars present means ± SD and each dot for independent image (n ≥ 8) from four distinct experiments. Statistical analysis was done using the Wilcoxon rank-sum test; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
- Figure 6. Single-cell analysis of IRF3 nuclear localization in HEV-infected HepG2/C3A cells.
HepG2/C3A cells expressing IRF3-GFP were electroporated with WT HEV RNA and fixed 2 d.p.e. after staining with viability stain (Zombie aqua), and were then stained for ORF2 (APC) and nucleus (Hoechst). Single-cell images of viable ORF2+ cells were obtained and categorized according to the localization of IRF3 and ORF2. (A) Cells with translocation of ORF2 and IRF3 into the nucleus are represented by TrORF2+ and TrIRF3+. Cells without translocation of ORF2 and IRF3 into the nucleus are represented by TrORF2− and TrIRF3−. (B) Percentage of HEV-replicating or ORF2+ cells with nuclear IRF3 translocation (ORF2+ TrIRF3+). (C) Percentage of ORF2 nuclear translocation (TrORF2+) among TrIRF3+ cells. Bars represent means ± SD, and each dot represents one independent experiment (n = 4). Statistical analysis was performed using a two-sided Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
Supplementary Materials
Table S1. Primer used for RT–qPCR.
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